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Paul Russell, Kaitlin Murphy, Joshua A. Wood, Clayton T. McKee, Christopher J. Murphy; Crossed Linked Actin Networks are Formed in Human Trabecular Meshwork Cells after treatment with Latrunculin B. Invest. Ophthalmol. Vis. Sci. 2012;53(14):6946.
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Crossed Linked Actin Networks (CLANs) have been reported in cells in the trabecular meshwork of individuals with glaucoma. CLANs have also been reported in approximately 5% of confluent human trabecular meshwork (HTM) cells in vitro. The number of cells with CLANs increases dramatically after treatment of HTM cells with dexamethasone for 10 days. Latrunculin B (Lat B), a compound that disrupts the actin cytoskeleton of cells, reduces intraocular pressure and is in development as a treatment for glaucoma. Our lab has determined that approximately 90 minutes after treatment of HTM cells with Lat B, the cell stiffness is increased above pre-treatment values and returns to pre-treatment values after six hours. We wished to determine if CLANs were formed in these cells as a result of cytoskeleton rearrangement potentially contributing to the dynamic changes observed in cell stiffness.
Cells at low cell density were treated with 2 micromolar Lat B for 30 minutes or with vehicle (DMSO) alone. Cells were fixed after removal of the drug at 30, 50, 90, 120 and 270 minutes and stained for actin using fluorescently labeled phalloidin and DAPI. Cells were imaged and analyzed for the presence of CLANs.
Five percent of the HTM cells prior to Lat B treatment were positive for CLANs. After Lat B treatment, the number of cells containing CLANs increased with time and reached 40% at 90 minutes. The number of cells with CLANs after 270 minutes was higher (12%) than the number in the control cells. The number of CLANs in cells treated with just vehicle alone remained at approximately 5% throughout the experiment.
This is the first demonstration that CLANs can be present in non-confluent single HTM cells without extended treatment with dexamethasone. The percent of CLANs in HTM cells was similar to that reported after dexamethasone treatment. The peak of cells containing CLANs occurred at a similar time as the increase in cell stiffness measured in living HTM cells after Lat B treatment. This suggests that the increase in cell stiffness after Lat B treatment is correlated to the presence of CLANs. This represents the first demonstration that CLANs and CLAN formation can be studied in single cells in a short time frame.
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